V. P. Whittaker

THE MORPHOLOGY AND ACETYLCHOLINE CONTENT OF ISOLATED CEREBRAL CORTICAL SYNAPTIC VESICLES

WHEN brain tissue is homogenized under carefully controlled conditions, a high proportion of the presynaptic nerve terminals are torn away from their attachments to form discrete particles for which the term ‘synaptosomes’ has recently been adopted (WHITTAKER, MICHAELSON and KIRKLAND, 1964). These particles may be separated from other subcellular brain particles by differential and density gradient centrifuging. They retain all the structural features of the nerve endings, together with their content of transmitter substances. On suspension in hypotonic media, a proportion of them swell and burst, discharging the contents of their cytoplasm. A simple density gradient procedure enables the various components of the synaptosome-soluble cytoplasmic constituents, synaptic vesicles, external membranes and intra-terminal mitochondriato be separated from the disrupted synaptosomes in relatively pure form, thus enabling the compartmentation of transmitter substances and other constituents of the nerve ending to be determined (WHITTAKER et al., 1963, 1964). Synaptosomes were first prepared by HEBB and WHITTAKER (1958) and first identified as such by GRAY and WHITTAKER (1960, 1962; see also WHITTAKER, 1960). The effect of hypotonic disruption was studied by WHITTAKER (1959, 1961), JOHNSTON and WHITTAKER (1963), DE ROBERTIS, RODRIGUEZ DE LORES ARNAIZ, SALGANICOFF, PELLEGRINO DE IRALDI and ZIEHER (1963) and WHITTAKER et al. (1964) (definitive papers only are quoted). The subject has been comprehensively reviewed by WHITTAKER (1964 a, b). This paper describes some further observations which have been made on the morphology and acetylcholine content of pure preparations of isolated synaptic vesicles. In a study of the properties of synaptosomes isolated from various areas of the central nervous system, regional differences in stability were observed during exposure to hypotonic conditions. Synaptosomes from the cerebral cortex were found to be more readily disrupted than those from some other areas and to give a higher yield of synaptic vesicles. Accordingly, cortical tissue was used as the source of synaptic vesicles in the present study. Methods have been devised for determining the average number of acetylcholine molecules/vesicle and the findings are discussed in relation to the problem of the quantized release of acetylcholine at cholinergic nerve endings. Preliminary accounts of this work have been given by SHERIDAN and WHITTAKER (1964) and WHITTAKER (1 964 c).

The preparation and characterization of synaptic vesicles of high purity

Very pure preparations of synaptic vesicles have been obtained from guinea pig cerebral cortex and from the electromotor synapses of Torpedo marmorata by density gradient centrifugation in a zonal rotor followed by chromatography on columns of glass beads of controlled pore size. Markers for soluble cytoplasm (lactate dehydrogenase), plasma and endoplasmic membranes membranes (Na-K-ATPase; acetylcholinesterase, NADPH-cytochrome c reductase], mitochondrial membranes [cytochrome oxidase] and lysosomes [acid phosphatase] were used to assess contamination and were undetectable. The only enzymes detected in the highly purified preparations from guinea pig cerebral cortex were Mg- and Ca-activated ATPases, but their content relative to acetylcholine fell on chromatography suggesting that they may be constituents of non-cholinergic vesicles. Lipids analyses of the highly purified vesicles confirmed earlier results and showed that glycolipids and lysolecithin are present in negligible amounts; this suggests that lysolecithin is not required for exocytosis of synaptic vesicles. A discussion of the probable limiting concentration of acetycholine in cerebral cortical vesicles derived solely from cholinergic terminals suggests that from 13 to 56% of the vesicles isolated are cholinergic, depending on the assumptions made.

VESICULAR STORAGE AND RELEASE OF ACETYLCHOLINE IN TORPEDO ELECTROPLAQUE SYNAPSES

Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [3H]acetate as a precursor of ACh. Little vesicular [3H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post‐stimulation recovery the newly synthesized [3H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [3H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.

The lipid and protein content of cholinergic synaptic vesicles from the electric organ of Torpedo marmorata purified to constant composition: implications for vesicle structure.

The lipid, protein, acetylcholine and ATP content of cholinergic synaptic vesicles isolated from the richly innervated electric organ of Torpedo marmorata and purified to constant composition has been determined. The number of vesicles present in the preparations has been estimated by quantitative electron microscopy and the mean composition of the vesicle deduced. The acetylcholine content of the purest preparations was considerably greater than that previously attained and reached a mean of 6.10 mmole/g of protein and 2.6 X 10(5) molecules/vesicle; the mean values, for all determinations, were 4.1 +/- S.E.M. 0.6 and 2.6 X 10(5) +/- S.E.M. 0.6 X 10(5) respectively. The lipid and protein content of the vesicle (about 140 and 80 ag/vesicle respectively) is relatively low, indicating a thin, lipid-rich membrane and a highly hydrated core of which not more than 1-2% can be occupied by protein. These findings are consistent with conclusions drawn from recent density determinations made at different osmotic pressures using penetrating and non-penetrating gradients.

DIFFERENT RECOVERY RATES OF THE ELECTROPHYSIOLOGICAL, BIOCHEMICAL AND MORPHOLOGICAL PARAMETERS IN THE CHOLINERGIC SYNAPSES OF THE TORPEDO ELECTRIC ORGAN AFTER STIMULATION

—During stimulation there occurred a decay in electrical response, vesicular acetylcholine, ATP and nucleotide as well as a loss of vesicle number and a decrease in vesicle diameter in the electric organ of Torpedo. These alterations were re‐established during a subsequent recovery period. The different parameters recovered at different rates. Firstly, electrical response to single pulses recovered to prestimulation values within about 5 h. Vesicle number and diameter as well as bouton size were found to be re‐established fully after 24 h. The newly formed vesicles appeared to be empty as vesicular acetylcholine, ATP and total nucleotide recovered much more slowly and were back to control values after about three days. Acetylcholine reappeared more quickly in the vesicles than ATP. Only after recovery of the vesicular pool of transmitter and ATP did the electric organ regain full stability of the electric discharge pattern on restimulation.

Immunohistochemical localization of cholinergic nerve terminals

SummaryMost of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.

Homocholine and acetylhomocholine: False transmitters in the cholinergic electromotor system of Torpedo

Abstract The subcellular distribution and acetylation of the choline analogue, N, N,N-trimethyl-N-3-hydroxypropylamine (homocholine) was studied using the electromotor system of Torpedo marmorata. Both choline and homocholine were transported into nerve terminals by the same high-affinity uptake mechanism (KT 2.0 and 3.5 μ m respectively), and became at least partly acetylated when blocks of electric tissue were perfused with [14C]choline and [3H]homocholine. Uptake of newly synthesized acetylcholine and acetylhomocholine into synaptic vesicles was enhanced by low-frequency stimulation of the block, and occurred into a metabolically active sub-fraction. Subsequent high frequency stimulation elicited the release of both14C and3H, in the same ratio as the two labels were isolated from the vesicle fraction. In other experiments it was shown that unacetylated homocholine was also released by stimulation, and found to be incorporated into vesicles in the same ratio to acetylhomocholine as it appeared in the perfusate. We conclude (a) that homocholine is not only a precursor to a cholinergic false transmitter (acetylhomocholine), but also qualifies as such, itself; (b) that uptake into vesicles under these conditions is not absolutely specific for acetylcholine and that prior acetylation is not an essential pre-requisite; and (c) that both acetylcholine and the two false transmitters are released from a vesicular compartment upon stimulation.

Metal ion content of cholinergic synaptic vesicles isolated from the electric organ of Torpedo: Effect of stimulation-induced transmitter release

Abstract Cholinergic synaptic vesicles were isolated from the Torpedo electric organ by a combination of differential and density gradient centrifugation. Iso-osmolar solutions of glycine or NaCl were used as homogenization and preparation media. The metal content of intact tissue and subcellular fractions were determined by atomic absorption spectroscopy. In the synaptic vesicle fraction ratios of metals to acetylcholine (g atom/mol) were: Na, 0.30; K, 0.10; Mg, 0.07; Ca, 0.28. Filtration of isolated vesicles revealed that the strength of metal binding depends on the ionic potential of the metal cation. Thus alkali metal ions are bound to synaptic vesicles less tightly than alkaline earth metals. Incubation of vesicles with elevated levels of NaCl led to a partial exchange of Na with K but external concentrations of CaCl 2 in the physiological range were without effect on vesicle metal ion content. Stimulation of the electric organ in vivo (5000 impulses, 5 Hz) caused a depletion of the acetylcholine and adenosine 5′-triphsophate content of the vesicles whereas the levels of metal ions were increased. It is suggested that the release of acetylcholine from synaptic vesicles exposes free negative charges to which extracellular metal cations can bind in ion exchange.

Changes in the biochemical and biophysical parameters of cholinergic synaptic vesicles on transmitter release and during a subsequent period of rest

Abstract Synaptic vesicles were isolated on sucrose density gradients from perfused blocks of Torpedo electric organ under varying experimental conditions. Newly synthesized acetylcholine was labelled with [ 3 H]acetate in order to distinguish synaptic vesicles which had been recycled during stimulation-induced transmitter release and, in consequence, had become smaller and denser from the larger, lighter vesicles characteristic of unstimulated tissue. After 1800 pulses at 0.1 Hz, the density of these smaller vesicles increased from a pre-stimulation value of 1.056 g.ml −1 to 1.067 g.ml −1 , whereas their water space (measured as the space occupied by the permeant solute glycerol), decreased by 34% from 65% to 43% of vesicle volume. During a subsequent 12 h rest period, these changes were partially reversed; water space returned to 52% of control and this change was highly correlated with a decrease in vesicle density and an increase in vesicular acetylcholine. The diameter of vesicles in whole tissue sections showed corresponding changes. An additional 12 h rest period did not lead to further significant recovery, suggesting that the preparation had a limited suitability for following long term processes depending on normal energy metabolism. The results can be explained on the assumption that when vesicles reform after releasing transmitter their core has a lower osmotic pressure than that of fully loaded vesicles. Reloading is accompanied by osmotically induced rehydration.

Separation of Recycling and Reserve Synaptic Vesicles from Cholinergic Nerve Terminals of the Myenteric Plexus of Guinea Pig Ileum

Acetylcholine‐rich synaptic vesicles were isolated from myenteric plexus‐longitudinal muscle strips derived from the guinea pig ileum by the method of Dowe, Kilbinger, and Whittaker [J. Neurochem.35, 993–1003 (1980)] using either unstimulated preparations or preparations field‐stimulated at 1 Hz for 10 min using pulses of 1 ms duration and 10 V ˙ cm−1 intensity. The organ bath contained either tetradeuterated (d4) choline (50 μM) or [3H]acetate (2 μCi ˙ ml−1); d4 acetylcholine was measured by gas chromatography‐mass spectrometry. As with Torpedo electromotor cholinergic vesicle preparations made under similar conditions the distribution of newly synthesized (d4 or [3H]) acetylcholine in the zonal gradient from stimulated preparations was not identical with that of endogenous (d0, [1H]) acetylcholine, but corresponded to a subpopulation of denser vesicles (equivalent to the VP2 fraction from Torpedo) that had preferentially taken up newly synthesized transmitter. The density difference between the reserve (VP1) and recycling (VP2) vesicles was less than that observed in Torpedo but this smaller difference can be accounted for theoretically by the difference in size between the vesicles of the two tissues. At rest, a lesser incorporation of labelled acetylcholine into the vesicle fraction was observed, and the peaks of endogenous and newly synthesized acetylcholine coincided. Stimulation in the absence of label followed by addition of label did not lead to incorporation of labelled acetylcholine, suggesting that the synthesis and storage of acetylcholine in this preparation and its recovery from stimulation is much more rapid than in Torpedo.